Method for improving the growth and colorimetric detection of bacteria, yeasts, fungi or cocci

ABSTRACT

The present invention relates to a method for improving the growth and detection of bacteria, yeasts, fungi or cocci, by adding sterile-filtered yeast extract and/or p-iodonitrotetrazolium violet to the culture medium.

This application is a division of U.S. Ser. No. 08/889,927, filed Jan. 8, 1997, now U.S. Pat. No. 6,168,930 which claims priority to German Application 196 02 345.9 filed Jan. 24, 1996.

The present invention relates to a method for improving the growth and the detection of bacteria, yeasts, fungi, or cocci, by adding to the culture medium sterile-filtered yeast extract and/or p-iodonitrotetrazolium violet.

The method is especially suited for the detection of mycobacteria or germs under stress conditions, such as airborne germs after the stress of desiccation in the air.

Special embodiments of the method of the invention are described hereinbelow.

The growth of bacteria, yeasts, fungi, cocci and germs is performed commonly on culture media known for the purpose. Their detection can then be performed by colorimetric methods using appropriate indicators.

For bacteria, cocci and other germs, nutrient mediums are generally used, such as tryptic soy agar or broth; yeasts and fungi can be cultured, for example, on Sabouraud agar, broth or RMPI 1640 broth. Common methods for mycobacteria are performed on a basis of egg or egg jelly, such as Löwenstein-Jensen or Stonebrinlk or agar media such as Middlebrook 7H10, 7H11, or fluid media such as Middlebrook 7H9 or Kirchner medium with 10% horse serum (DIN 58 943-3, Manual of Clinical Microbiology, 6th ed., 414-416).

For growth detection, the evaluation of C¹⁴ palmitic acid can be used, which releases ¹⁴CO₂ (Bactec, Manual of Clinical Microbiology, 6th ed., 415); also oxygen consumption in the medium which is indicated by a fluorescent indicator (EP-A 0 509 781 Al, commercial name of the product MGIT) or by barometric measurement (ESP Automat für Blutkultur und Mykobalkterien, Difco) and by redox indicators such as resazurine/methylene blue (DE 4 316 394) or tetrazolium chloride (Deutsche Medizinische Wochenschrift 75, 1471 (1995)).

The growth of bacteria takes place relatively slowly. Mycobacteria on solid media require about 2-6 weeks, depending on the mycobacteria used for inoculation, and in liquid media 1-3 weeks. Growth detection with additional apparatus is very expensive, as is also the disposal of the radioactive waste. In known colorimetric redox indicators such as resazurine/methylene blue or tetrazolium chloride, the effect is seen that, for example, clinical strains of Mycobacteria tuberculosis are inhibited at low germ counts and do not grow satisfactorily except in high germ counts. However, even low germ counts have to be detected reliably in clinical test material. Furthermore, these indicators are toxic in the large amounts which are needed for low germ counts. Reducing the amount of these colorimetric redox indicators reduces the toxicity, but the result is that clinical isolates, such as those of Mycobacterium tuberculosis, do grow, but they are no longer colored, i.e., the desired colorimetric detection is not accomplished.

In the isolation and detection of airborne gents of various kinds, such as bacteria, cocci, yeasts, fungi and spores, the following problems occur: airborne germs are stressed by desiccation in the air. Moreover, the media for the detection of the germs are often gamma-sterilized, which again means stress for the medium and therefore leads to poorer growth of the germs to be detected. Both factors lead to the fact that germs of certain kinds, especially gram positive bacteria, and cocci, can no longer be reliably detected in the usual manner (colorimetry with resazurine).

The present invention is therefore addressed to the problem of developing a method by which the growth of bacteria, fungi, yeasts, cocci, can be improved and the detection of the targeted species can be performed reliably and at low cost.

This problem is solved according to the invention by adding to the culture medium sterile-filtered yeast extract and/or p-iodonitrotetrazolium violet.

It was found surprisingly that by the addition of sterile-filtered yeast extract or p-iodonitrotetrazolium violet the growth of bacteria, fungi, yeasts, cocci, can be accelerated. If the redox indicator p-iodonitrotetrazolium violet (INT) is added, the growth of the species under study can be detected simply by colorimetry, while the intensity of calorimetric detection with both additives is synergically increased by the addition of the sterile-filtered yeast extract.

The method of the invention is especially suited to the detection of mycobacteria, and especially clinical Mycobacterium tuberculosis isolates which are growing on various media such as 7H9 broth, 7H12, 7H9 with OADC ((oleic acid, albumin, dextrose, catalase, for Trademark, see Difco Manual) and PANTA (polymyxin, amphotericin B, nalidixic acid, trimethoprim, azlocillin) in the MGIT system Mycobacteria Growth Indicator Tube); in Kirchner medium with horse serum, and on solid media such as Löwenstein Jensen.

Even bacteria, especially mycobacteria, damaged by long holding grow better with the additive according to the invention.

The time required for this purpose can be considerably reduced, and detection itself can be simplified.

For example, the sensitivity testing of mycobacteria, which otherwise is possible only within 1 week with the use of a radioactive substrate (¹⁴C palmitic acid in Becton Dickinson's Bactex 460 system), can be evaluated even visually within 5 to 7 days by measuring turbidity upon the addition of sterile filtered yeast extract to 7H9 broth, without the need for high-cost systems such as radioactivity detection with the disposal problems which they entail. Furthermore, by the use of a combination with colorimetric substrates, namely INT, the color intensity of the indicator is increased.

The addition made according to the invention is also especially suited for the detection of air-borne germs, because the increase obtained in the germ count by sterile filtered yeast extract results in more rapid growth generally and especially in faster growth than in media that are not gamma-sterilized. Thus the calorimetric detection of germs, especially gram negative bacteria, yeasts and fungi, becomes possible if INT is added as indicator. This also permits the gamma-sterilization of media, and the sterile-filtered yeast extract compensates the damage to the media as regards growth properties. Thus, germs grow on gamma-sterilized media with sterile-filtered yeast extract even slightly better than on conventional, non-gamma sterilized media.

Tryptic soy agar or other such nutrient media can be used as the basic medium.

The addition of sterile-filtered yeast extract according to the invention is performed preferably in liquid form in amounts of 0.5 to 10 g/l, especially 0.5-5 g/l, and 2-2.5 g/l is very especially preferred.

The indicator, iodonitrotetrazolium violet (INT) is added to the culture medium preferably in an amount of 1 to 30 mg/l, especially 5-20 mg/l, and especially 8-15 mg/l.

Furthermore, the growth accelerated according to the invention can be detected calorimetrically in other ways, for example by now adding a small amount of the resazurine/methylene blue system (cf. DE 43 16 394), since the detection of even lower germ counts is possible due to the accelerated growth. This system can be varied in a known manner according to the purpose of the detection; for example, tuberculostatic agents can be added in the sensitivity testing of mycobacteria if growth control is desired. Also, redox stabilizers can be added in this case. The indicators are present in conventional amounts such as 1-200 mg/l and 5-100 mg/l of nutrient medium.

Growth can also be accelerated in other commercial systems by the addition of sterile-filtered yeast extract, as shown in Example 10 below, with Bactec 12B with sterile-filtered yeast extract in comparison with Example 11 (commercial Bactec 12B system), and in accordance with Examples 6, 7 and 8 in MGIT with sterile-filtered yeast extract, and sterile-filtered yeast extract plus INT and INT in comparison with Example 5 in commercial MGIT of Becton Dickinson.

With the addition made according to the invention, it is thus possible in general to accelerate the growth of bacteria, yeasts, cocci and fungi, and in addition to avoid the use of expensive detection apparatus.

The invention will be further explained in the following examples.

I. EXAMPLES 1-12

The growth of Mycobacterium tuberculosis was tested on various known media (solid or liquid) in different amounts of inoculant. Media 2, 3, 4, 6, 7, 10 and 12 contain the addition, in accordance with the invention, of sterile-filtered yeast extracts (2, 3, 6, 7, 10, 12) and/or INT (3, 4, 7, 8).

Table I presents the results of this experiment. As it can be seen, a faster growth is achieved with the additive of the invention in specimens 2 and 3 in comparison with 1 (no additive), in specimens 6 and 7 (the invention) in comparison with 9 (comparative example), and in 12 (the invention) in comparison with 11 (comparative example). Furthermore, with the aid of the media containing INT in addition (3 and 7, yeast extract and INT) a more intense coloring is found in comparison with the corresponding media with INT oney (4 and 8).

Media for the Isolation of Mycobacteria (Examples 1 to 12) 3. Kirchner 1. Kirchner 2. Kirchner Medium with 4. Kirchner Medium Medium with yeast extract Medium normal yeast extract and INT with INT Disodiumhydrogenphosphate-12-hydrate 3.0 g 3.0 g 3.0 g 3.0 g Potassium dihydrogen phosphate 4.0 g 4.0 g 4.0 g 4.0 g Magnesium sulfate 0.6 g 0.6 g 0.6 g 0.6 g Sodium citrate 2.5 g 2.5 g 2.5 g 2.5 g L-Asparagine 0.1 g 0.1 g 0.1 g 0.1 g Potassium aspartate 4.0 g 4.0 g 4.0 g 4.0 g D-Alanine 2.0 g 2.0 g 2.0 2.0 g Sodium pyruvate 0.5 g 0.5 g 0.5 g 0.5 g Hemin (dissolved in 1 ml NaOH) 1.66 mg 1.66 mg 1.66 mg 1.66 mg Ammonium iron(III) sulfate 50 mg 50 mg 50 mg 50 mg Distilled water 900 ml 800 ml 800 ml 900 ml Glycerine autoclaved separately 20 ml 20 ml 20 ml 20 ml Horse serum 100 ml 100 ml 100 ml 100 ml (inactivated 1 h at 56° C.) Catalase solution 5 ml 5 ml 5 ml 5 ml (Boehringer Mannheim 106836) Ribonucleic acid solution 25 μg 25 μg 25 μg 25 μg Yeast extract, sterile-filtered — 2.5 g in 100 ml 2.5 g in 100 H₂O ml H₂O INT Solution (Sigma 9405), containing 625 mg/L) — — 24 ml 24 ml INT = p-iodonitrotetrazoliumviolet = (2-[- iodophenyl]-3-[4-nitrophenyl-5 phenyltetrazolium chloride

The first 12 components are autoclaved together, and the rest are added as sterile-filtered, sterile solutions (without autoclaving).

Yeast extract is available commercially. 5 g yeast extract is dissolved in 200 ml dist. water by stirring for 1 hour. The resulting solution is filtered over a prefilter, followed by a sterile-filtration using a 0.2 μm filter as final filter. The prefilter is a Nillex-AP50 prefilter from Nillipore

5. MGIT from Becton Dickinson 6. MGIT with sterile-filtered 4 ml 7H9 broth yeast extract in 0.4 ml water +OADC 4 ml 7H9 broth +PANTA +OADC all components from Becton +PANTA Dickinson +10 mg yeast extract sterile- 7. MGIT with sterile-filtered yeast filtered in 0.4 ml water extract and INT 8. MGIT with INT 4 ml 7H9 broth 4 ml 7H9 broth +OADC +OADC +PANTA +PANTA +10 mg yeast extract sterile- +0.100 ml INT solution filtered in 0.4 ml water (from Sigma, containing +0.100 ml INT solution 625 mg/L) (from Sigma, containing 10. Bactec 12B 625 mg/L) Becton Dickinson's broth 9. Bactec 12B (basing on 7H9) Becton Dickinson's broth 4 ml (basing on 7H9) +PANTA 4 ml +10 mg yeast extract sterile- +PANTA filtered in 0.4 ml H₂O 11. Löwenstein Jensen medium 12. Löwenstein Jensen medium with sterile-filtered extract I Salt solution Potassium hydrogen 2.4 g 2.4 g phosphate Magnesium sulfate-7 0.24 g 0.24 g hydrate Trimagnesiumdi- 0.6 g 0.6 g citrate-14-hydrate L-asparagine mono- 3.6 g 3.6 g hydrate glycerine 84-87% solution 12.0 ml 12.0 ml V/N Distilled water 600 ml 350 ml II Malachite Green Solution Malachite green 2.0 g 2.0 g Dist water to make 100.0 to make 100. ml ml III Potato flour emulsion Salt solution from I 600 ml 350 ml Malachite solution 20 ml 20 ml from II Potato flour 30 g 30 g Emulsion sterilized 30 min at 112° C. IV 1000 ml 1000 1000. ml of 20-25 eggs ml, mix homogenized with mix homogenized with 370 ml of III + 620 ml of III 150 ml H₂O containing 4.0 g yeast extract sterile-flitered

TABLE I Growth of Mycobacterium tuberculosis inoculated in different amounts on the specified media Growth on medium No. in days Amount of 1 2 3 4 5* 6* 7* 8* 11 12 9 10 inoculum Std. Inv Invention Invention Std. Inv Invention Invention Std Inv. Std. Inv. M. Tuberculosis H37RV Mcfarland 0.5 x 10⁻² 7 5 5 purple 7 purple 6 5 5 purple 6 purple 10 8 5 4 10⁻³ 11 8 8 purple 10 purple 9 7 7 purple 8 purple 14 11 6 5 10⁻⁴ 14 11 11 dark 12 purple 12 10 10 dark 11 purple 18 14 9 7 purple purple 10⁻⁵ 16 12 12 dark 15 purple 14 12 12 dark 13 purple 22 17 11 10 purple purple 10⁻⁴ 23 16 15 dark 10 purple 17 14 14 dark 15 purple 28 20 13 12 purple purple 10⁻⁷ 28 19 19 purple 24 purple 20 18 18 dark 19 purple 33 25 17 16 purple Clinical M. Tuberculosis 1 Mcfarland 0.5 x 10⁻² 9 6 6 purple 6 purple 8 6 6 purple 7 purple 11 9 5 4 10⁻³ 13 9 8 purple 12 purple 12 9 9 purple 11 purple 15 13 9 7 10⁻⁴ 17 11 10 dark 16 purple 16 12 11 dark 16 purple 20 16 11 10 purple purple 10⁻⁵ 22 13 13 dark 20 purple 21 14 14 dark 20 purple 25 20 14 12 purple purple 10⁻⁶ 26 17 16 dark 25 purple 25 18 17 dark 25 purple 30 24 17 15 purple purple 10⁻⁷ 32 21 20 dark 30 purple 30 20 20 dark 29 purple 36 28 19 18 purple purple Clinical M. tuberculosis 15 Mcfarland 0.5 x 10⁻² 8 5 5 purple 8 purple 6 5 5 purple 6 purple 11 9 5 4 10⁻³ 12 8 8 purple 12 purple 9 8 8 purple 9 purple 14 12 7 6 10⁻⁴ 15 11 11 dark 15 purple 12 11 11 dark 12 purple 18 14 10 9 purple purple 10⁻⁵ 22 13 13 dark 21 purple 14 12 12 dark 14 purple 25 19 12 11 purple purple 10⁻⁶ 26 17 16 dark 25 purple 18 16 15 dark 17 purple 30 23 15 14 purple purple 10⁻⁷ 29 20 19 dark 29 purple 22 19 18 dark 20 purple 35 27 18 16 purple purple *In 5 and 6, evaluation by fluorescence in accordance with instructions on MGIT In 7 and 8, evaluation by fluorescence in accordance with instructions on MGIT and additional occurence of purple colonies (due to INT)

II. EXAMPLES 13-15

In these examples, sensitivity testing based on the method described in DIN 58942, Part 8, for the detection of mycobacteria was employed in the case of the fluid media 13, 14 and 15 to 15C. The evaluation was performed by the finding of turbidity in the control (and in the case of resistance, also by turbidity) in the case of media 13 and 14. In media 15 to 15C, resistance and growth were evaluated by the occurrence of purple-colored colonies.

Use was made of 7H9 medium with ADC enrichment (standard) and, for the invention, 7H9 medium with ADC enrichment plus sterile-filtered yeast extract, as well as 7H9 medium with ADC enrichment plus sterile-filtered yeast extract plus INT.

DIN 58942, Part 8 is essentially the proportion method as described by Leonid B. Heifets in Drug susceptibility in the Chemotherapy of Nycobacterial Infections, Boca Raton, Fla. (CRC Press) 1991, page 89-121. It is used as one of the standard methods at the National Jewish Hospital in Denver (Colo.) by Mr. Heifets.

13 Middlebrook 7H9 14 with addition of 15 with addition of broth 4,7 g/l sterile-filtered yeast sterile-filtered yeast autoclaved extract extract and INT Middlebrook 7H9 +ADC enrichmnent Middlebrook 7H9 broth 4.7 g/l 100 ml to 900 ml broth 4.7 g/l autoclaved 7H9 base medium autoclaved 800 ml 7H9 base (per instructions of 800 ml 7H9 base medium the medium medium 100 ml ADC manufacturer, Difco) 100 ml ADC enrichment enrichment 100 ml H₂O (Standard) 100 ml H₂O, containing 1.5 g containing 2.5 g sterile-filtered yeast sterile-filtered yeast extract extract 24 ml INT solution 15A differing from (The invention) 15 as follows: 100 ml H₂O containing 3.0 g sterile-filtered yeast extract. 15B differing from 15 as follows: 100 ml H₂O containing 2.0 g sterile-filtered yeast extract; 15C differing from 15 as follows: 100 ml H₂O containing 0.5 sterile-filtered yeast extract (The invention)

The results are reflected in the following Table II.

TABLE II MIC* in Readable MIC after days in Medium Strain Antituberculo-static μg/ml 13 std. 14 inv. 15 inv. 15A 15B 15C M.tb. H₃₇ R_(v) Rifampicin (RMP) 2.0 9 5 5 5 5 6 Clin. M.tb 1 Rifampicin (RMP) 1.0 11 7 7 7 7 8 Clin. M.tb 15 Rifampicin (RMP) 2.0 9 5 5 5 5 6 M.tb. H₃₇ R_(v) Isoniacid (INA) 0.06 9 5 5 5 5 6 Clin. M.tb 1 Isoniacid (INA) 0.03 11 7 7 7 7 8 Clin. M.tb 15 Isoniacid (INA) 0.03 9 5 5 5 5 6 M.tb. H₃₇ R_(v) Ethambutol (EMB) 0.5 9 5 5 5 5 6 Clin. M.tb 1 Ethambutol (EMB) 0.25 11 7 7 7 7 8 Clin. M.tb 15 Ethambutol (EMB) 0.5 9 5 5 5 5 6 *MIC = minimum inhibiting concentration

As it is apparent from the above, specimens 14 and 15 are evaluated more rapidly with the additive according to the invention than in the standard medium 13.

Moreover, calorimetric detection is possible with the addition of INT in media 15 to 15C. Examples 15A-15C show different amounts of yeast extract added.

III. EXAMPLES 16-18

In the following Examples 16-18 are listed media with (Examples 17, 18) and without (Example 16) the additive according to the invention, which were used with (16A, 17A) and without (16, 17, 18) previous gamma sterilization (gamma rays of 20-30 Kilogray) as growth media for different airborne germs listed in Table III.

16. Tryptic soy agar for germ 17. Tryptic soy agar for germ counting, counting. with sterile-filtered yeast extract 15 g 15 g Pancreatic digest of casein 5 g 5 g soybean peptone 5 g 5 g NaCl 0.7 g 0.7 g lecithin 7 g 7 g polysorbate 80 17 g 17 g agar 900 ml H₂O 1000 ml H₂O The mixture is autoclaved The mixture is autoclaved Add 100 ml sterile H₂O containing 2.5 g of sterile-filtered yeast extract 18. Tryptic soy agar for germ counting with sterile-filtered yeast extract and INT 15 g pancreatic digest of casein 5 g soy bean peptone 5 g NaCl 0.7 g lecithin 7 g polysorbate 80 17 g agar 900 ml H₂O The mixture is autoclaved Add 100 ml sterile H₂O containing 2.5 g sterile- filtered yeast extract.

As it is apparent from Table III, medium 17 (inv.) shows a substantial improvement in the number of re-found colonies over 16 (std.)

It can also be seen that the number of re-found colonies diminishes due to the stress of the gamma radiation (media 16A, 17A) as compared with the non-irradiated media (16, 17).

If 17A (sterile-filtered, plus additive according to the invention) is compared with 16 (standard), it is seen that a gamma-irradiated medium which still contains sterile-filtered yeast extract still provides at least the same, but sometimes better results than a non-irradiated normal medium.

TABLE III Strain and amount Growth on medium No., and number of colonies after 1 day inoculate 16 16A 17 17A 18 Bacillus subtilis 10³ 80 11 profuse 95 58 purple colonies 2 × 10² 10 1 52 20 0 purple colonies 50  1 0 16  4 0 purple colonies Staph. aureus ATCC 6538 10³ profuse 68 profuse profuse 116 purple colonies 2 × 10² 138  27 profuse 119  2 purple colonies 50 32 1 68 35 0 purple colonies Candida pseudotropicalis 10³ 17 2 52 21 48 purple colonies 2 × 10²  3 0 12 11 10 purple colonies 50  1 0  3  4 3 purple colonies Alteromonas putrefaciens 10³ profuse 59 profuse profuse profuse purple 2 × 10² 66 21 139  78 128 purple colonies 50 11 2 51 24 48 purple colonies

IV. CLINICAL EXPERIMENTS

With a different nutrient medium and a medium containing the addition according to the invention of sterile-filtered yeast extract and INT, clinical experiments were conducted with various specimen materials for the detection of mycobacterium tuberculosum.

The results are given in Table IV below.

TABLE IV Medium 3 in Löwenstein Conventional Specimen accord with the Bectec 12B Jensen Kirchner media material invention (standard) (standard) (standard) Sputum* positive after 18 negative negative negative days Sputum positive after 18 positive after 18 positive after 28 positive after 21 days days** days days Sputum positive after 15 positive after 15 positive after 21 positive after 18 days days** days days Fluid from lung positive after 18 negative negative negative tap days Bronchial positive after 20 negative negative negative washing days Sputum positive after 16 negative negative negative days *Treated patients, Tb diagnosis confirmed by preliminary tests **In the medium according to the invention the differentiation of the species with commercial DNA probes of gene specimen can be performed immediately. In the case of Bectec 12B another 3-4 days are required in order to achieve a growth index of 500 so as to be able to use these probes reliably.

Table IV shows that germs damaged and stressed by previous treatment can still be cultured in the medium according to the invention, whereas this is no longer the case in the media with which it is compared.

Furthermore, immediate processing with molecular-biological DNA probes is possible with the medium according to the invention on the same day on which the positive finding of growth is made. Consequently differentiation of species and an immediate finding is possible at the clinics, whereas in traditional systems it is necessary to wait longer in order to have sufficient germs for testing with commercial DNA probes.

As it is dearly apparent from these examples, the method of the invention leads to fast, reliable results as regards the growth and detection of many different species. 

What is claimed is:
 1. A method for accelerating a growth of bacteria or fungi in culture, said method comprising the following steps: a) adding to a culture medium one member but not both members selected from the group consisting of i) yeast extract which has been sterile filtered without autoclaving and ii) p-iodonitrotetrazoliumn violet; and b) growing and detecting bacteria or fungi in said culture medium; wherein the growth of said bacteria or fungi is accelerated as compared to the growth of bacteria or fungi of the same species which have not been subjected to said adding.
 2. The method according to claim 1, wherein said bacteria are cocci and said fungi are yeasts.
 3. The method according to claim 1, which is for growing and detecting mycobacteria.
 4. The method according to claim 3, which is for growing and detecting strains of Mycobacterium tuberculosis.
 5. The method according to claim 1, which is for growing and detecting bacteria or fungi under stress conditions.
 6. The method according to claim 1, wherein said culture medium is selected from the group consisting of: a) liquid media optionally comprising oleic acid, albumin, dextrose, catalase, polymyxin, amphotericin B, nalidixic acid, trimethoprim and azlocillin or optionally comprising horse extract; b) solid media based on egg or egg jelly; and c) tryptic soy agar.
 7. The method according to claim 1, which comprises using a resazurine/methylene blue indicator system as an indicator.
 8. A method for accelerating a growth of bacteria or fungi in culture, said method comprising the following steps: a) adding to a culture medium yeast extract which has been sterile filtered without autoclaving; and b) growing and detecting bacteria or fungi in said culture medium; wherein the growth of said bacteria or fungi is accelerated as compared to the growth of bacteria or fungi of the same species which have not been subjected to said adding.
 9. The method according to claim 8, which comprises adding 0.5 to 10 g/l of said yeast extract which has been sterile filtered without autoclaving to said culture medium.
 10. A method for accelerating a growth of bacteria or fungi in culture, said method comprising the following steps: a) adding to a culture medium p-iodonitrotetrazoliumn violet; and b) growing and detecting bacteria or fungi in said culture medium; wherein the growth of said bacteria or fungi is accelerated as compared to the growth of bacteria or fungi of the same species which have not been subjected to said adding.
 11. The method according to claim 10, which comprises adding 1 to 30 mg/l of p-iodonitrotetrazoliur violet to said culture medium. 